Acute Nickel Toxicity Responses of Labeo rohita and Cirrhinus mrigala

| The exposure of acute nickel toxicity (96-hr LC 50 and lethal concentrations) for two fish species viz. Labeo rohita and Cirrhinus mrigala was determined in this study. During metal stress trials both species were kept at constant temperature (32°C), pH (7) and hardness (250 mg L -1 ) of water. Physico-chemical parameters of test medium were monitored regularly. The observed mean LC 50 and lethal concentrations for Cirrhinus mrigala were 55.85 ± 2.84 and 128.44 ± 9.25 mg L -1 , respectively. On the other hand, for Labeo rohita , mean values of LC 50 and lethal concentrations were calculated as 56.42 ± 2.51 and 120.98 ± 7.18 mg L -1 , respectively. Cirrhinus mrigala had lower LC 50 but higher lethal dose. Two fish were different in size as well. A significant positive correlation of metallic ion concentrations was observed with sodium, potassium, carbon dioxide and electrical conductivity whereas an inverse relationship was obtained with dissolved O 2 for both Labeo rohita and Cirrhinus mrigala test mediums. It is concluded that acute concentration of nickel is lethal for both fish species with a significant difference of LC 50 and lethal concentrations among two fish species. Novelty Statement | The study is novel in recommending continuous monitoring of metal contamination in fish species to ensure the sustainability of this food for human consumption.

Although due to presence of various pollutants, consumption of fish may become a threat for human beings.These chemically metallic substances are highly dangerous even at very low concentrations.Heavy metals are essential components of earth crust that occurs naturally and also measured as natural elements of aquatic system with limited permissible concentration.Now due to intense agricultural and industrial activities, its level of concentration has been increased (Zhang et al., 2011;Martín et al., 2015;Munari et al., 2017).
Specific toxicity tests are performed to check out harmful effects of heavy metals on aquatic creature (Akter et al., 2008;Javed, 2013).It allows to establish a dose-response relationship to find adverse effects of contaminants on the aquatic fauna and discharge rate of pollutants in aquatic organisms (Association et al., 1915).Acute toxicity tests are performed to determine the impact of pollutants on aquatic systems during their life expectancy (Ebrahimpour et al., 2010;Javed, 2012), to analyze metal toxicity on fish biology ( Javed and Saeed, 2010), to monitor their ability of adaptation under toxic levels and to estimate various possible toxicity impacts on them (Abdul et al., 2009;Azmat et al., 2012).Low level of essential metals i.e. copper, nickel, zinc, chromium, iron and cobalt causes various infections while their high level may also cause toxicity (Sivaperumal et al., 2007;Abadi et al., 2015).Low concentration of nickel is less damaging when present in fresh water bodies and it may be causes any type of morphological change or any chromosomal aberration within cells (Magyarosy et al., 2002).Now, fresh water bodies contaminated with pesticides have become a major issue that effect aquatic organisms badly (Indirabai et al., 2010;Iqbal et al., 2012;Naz and Javed, 2012).Heavy metals toxicity in rivers have shown adverse effects on native fish fauna including carps such as Catla catla, Cirrhinus mrigala and Labeo rohita.(Rauf et al., 2009;Azmat et al., 2012).Although among all pollutants, metals are of vital concern due to their diverse impacts as it causes toxicity in fish (Abdul et al., 2009).Keeping in view of toxic effects of nickel on aquatic habitats, posing threats to the freshwater fish, the present investigation was conducted to study the response of Labeo rohita and Cirrhinus mrigala to acute (LC 50 and lethal) toxicity of nickel.

Materials and Methods
The current work was performed in Zoology Laboratory, The Govt.Sadiq College Women University Bahawalpur.To evaluate the acute toxicity effect of nickel on Labeo rohita and Cirrhinus mrigala, a 96-hr LC 50 and lethal concentrations trials were carried out at 7 pH, 32°C temperature and 250 mg L -1 hardness of water in 70 liters glass aquaria.Fish were allowed to adapt the laboratory conditions for 12 hours prior to start of the experiment.A desired amount of pure compound NiCl 2 was mixed with de-ionized water.Then required metal dilutions was prepared from the stock solution.Different fish used for 96-hr LC 50 as well as for lethal toxicity tests were mentioned below (Table 1).For each test concentration, 20 fish of each species were kept in each glass aquarium for a period of 96-h.To prevent the fish from stress, metal concentration of each aquarium was gradually increased, 50% test concentration maintained in 3.5 hours along with full toxicant concentration in 7 hours.All test mediums were provided with constant air through air pump installed in each aquarium.Metal concentration for each fish species varied from zero with an addition of 0.05 and 5 mg L -1 (as a total concentration) for lower and higher concentrations.Fish mortality and physico-chemical variables (temperature, pH, total hardness, dissolved oxygen, total ammonia, sodium, potassium and carbon-dioxide) were recorded at 12 hours interval during 96 hours for each experimental trial (APHA, 1998).The experiment was ended after completion of 96-h of experiment.There were three replications for each experiment.The number of dead fish were counted during the experiment.

Statistical analysis
The complete data obtained after experiment was then statistically analyzed using micro-computer according to Steel et al. (1996).To check the statistical variations among different variables, Analysis of Variance (ANOVA) and Duncan's Multiple Range tests were carried out.In addition, correlation was also used to find associations among different factors.Probit Analysis Method (Hamilton et al., 1977) was used to analyze data on percent fish mortality that was attained during 96-h LC 50 and lethal concentration experiments.

Results and Discussion
An experiment was performed for 84-day age groups of two fish species viz.Cirrhinus mrigala and Labeo rohita.Separate experimental tests were performed for their sensitivity towards nickel LC 50 and lethal concentrations with 95% confidence interval.To determine their 96-hr LC 50 and lethal responses, both species were exposed to variable water-borne nickel concentrations.Mortality data was analyzed through Probit analysis (Table 2).The obtained mean values of 96-hr Ni LC 50 for Labeo rohita were as 56.42 ± 2.51 mg L -1 with confidence interval range of 51.45 -61.52 mg L -1 whereas its mean lethal concentration was monitored as 120.98 ± 7.18 mg L -1 among confidence interval of 109.09-138.35mg L -1 .High significant results were shown by probability graph of regression coefficient which demonstrates the fillness of regression line (Figure 1).At 95 percent confidence interval, normal distribution of data showed the mean Cirrhinus mrigala LC 50 value as 55.85 ± 2.84 mg L -1 among confidence interval range of 50.37 -61.81 mg L -1 whereas the mean lethal concentration was obtained as 128.44 ± 9.25 mg L -1 with confidence intervals of 113.47 -151.57mg L -1 (Figure 2).Statistically different responses (i.e.p ˂ 0.05) were observed for both fish species.Significantly more sensitive response for 96hr LC 50 was shown by Cirrhinus mrigala and significant low lethal response was shown by Labeo rohita (Table 2).Complete data concerned with correlation coefficients along with physico-chemical variables and nickel exposure concentrations of test mediums meant for both fish species was mentioned in Tables 3 and 4. A significant positive correlation of metallic ion concentrations was found with CO 2 , sodium, potassium, electrical conductivity and total ammonia.However, an inverse relationship was obtained with dissolved O 2 of test mediums, suggesting that different physio-chemical parameters may have significant impact on the obtained results of the experiment hence, their value must be kept on record throughout the experimental duration to keep avoid the unwanted impact of these parameters on actual experimental variables.Among different fish species, the acute toxicity (lethal concentration and 96-h LC 50 ) of metals may be varying.A fish may be highly sensitive to a specific metal but at the same time that fish may show high resistant to another metal (Biuki et al., 2010).Due to variable physico-chemical features of metals, the toxicity level of different metals may fluctuate significantly (Azmat et al., 2012).Metals can penetrate into the body through different ways e.g.gills, orally intake of water, foodstuff, non-food particles and skin.Basically, metals are absorbed and transported through blood to any storage organ such as liver for metals transformation or for bioaccumulation in different fish organs (Nussey, 2000;Rauf et al., 2009).Fish skin comes in direct contact with metal ions and usually consumed with muscles so it has more chances of metal contamination (Yousafzai and Shakoori, 2006).Variations in the rate of metals accumulation among different species are species specific (Giguère et al., 2004).An exposure to waterborne heavy metals causes an observable hypersensitivity in fish ( Javed, 2012).); Temp= Temperature ( O C); pH; T.H.= Total hardness (mg L -1 ); T.NH 3 =Total ammonia (mg L -1 ); D.O= Dissolved oxygen (mg L -1 ); CO 2 = Carbon Dioxide; E.C.= Electrical Conductivity (mS cm -1 ); Na= Sodium (mg L -1 ); K= Potassium (mg L -1 ); Ca= Calcium (mg L -1 ); Mg= Magnesium (mg L -1 ).
Current study indicated that 96-hr LC 50 nickel concentration varied significantly among the two fish species as Labeo rohita and Cirrhinus mrigala.Cirrhinus mrigala has lower 96-hr LC 50 (55.85± 2.84 mg L -1 ) than Labeo rohita (56.42 ± 2.51 mg L -1) .On the other hand, Labeo rohita was considerably more sensitive for lethal concentrations of nickel (120.98 ± 7.18 mg L -1 ) than Cirrhina mrigala (128.44 ± 9.25 mg L -1) .Kousar et al. (2020) examined three fish species viz.Catla catla, Labeo rohita and Cirrhinus mrigala which were exposed to chronic sub-lethal levels of nickel concentration of 70.40, 71.99 and 79.9 mg L -1 under the duration of 60 days of growth trial.The impact of nickel stress at 10, 20, 30 and 40 mgL -1 concentrations on Channa punctatus was observed for 30 days of trial (Desai et al., 2002).Kousar and Javed (2014) also determined the acute toxicity and bioaccumulation patterns of heavy metals (As, Ni and Zn) in Labeo rohita, Cirrhinus mrigala, Ctenopharyngodon idella and Catla catla.The order of metal accumulation was Zn ˃ Ni ˃ As.Compared with other fish species, Cirrhinus mrigala had significant ability towards Ni and Zn accumulation as 146.8 ± 149.1 µg g -1 and 243.0 ± 190.5 µg g -1 , respectively.(Naz and Javed, 2012) described the acute toxicity of metal mixtures (Fe, Zn, Pb, Ni and Mn) in Catla catla, Labeo rohita and Cirrhinus mrigala and observed high toxicity to fish in terms of 96 hr LC 50 .According to Yousafzai et al. (2010), the order of metal accumulation in L. dyocheilus muscles was Zn ˃ Cr ˃ Cu ˃ Pb ˃ Ni ˃ Cd.During present study, significant differences were observed between LC 50 and lethal concentrations of nickel for Labeo rohita and Cirrhinus mrigala.Mean nickel concentration LC 50 for Labeo rohita and Cirrhinus mrigala was 56.42 ± 2.51 mg L -1 and 55.85 ± 2.84 mg L -1 , respectively.On the other hand, the calculated mean of lethal concentration of nickel was 120.98 ± mg L -1 and 128.44 ± 9.25 mg L -1 for Labeo rohita and Cirrhinus mrigala, respectively.Results showed that Cirrhinus mrigala has lower LC 50 but higher lethal dose compared with Labeo rohita.The correlation of physio-chemical parameters to the metal toxicity and its bioaccumulation is well documented.In our study, except for dissolved oxygen, all studied parameters like CO 2 , sodium, potassium, electrical conductivity and total ammonia were directly proportional to the metal toxicity.Similar results were reported earlier stating that these physico-chemical parameters causes increase in metal toxicity for fish (Azmat et al., 2016).Yaqub and Javed (2012) also concluded that physiochemical parameters of fish aquaria have significant effect on heavy metal toxicity studied in these media.Physicochemical parameters such as, calcium, sodium, dissolved organic matter, and pH are reported to impact metal toxicity of different heavy metals to numerous aquatic organisms, like fish (Niyogi and Wood, 2004).Size and age of fish species also have significant effect on metal accumulation and variations shown in the results.

Conclusions and Recommendations
The current study concluded that nickel is a toxic metal for both the studied fish species when these are exposed to acute concentration of the metal in water bodies.While the LC 50 of Labeo rohita is higher than Cirrhinus mrigala, the lethal concentration of nickel for Cirrhinus mrigala is slightly higher as compared to the Labeo rohita.This study further concluded that physico-chemical parameters have significant impact on acute toxicity of the metal.Hence, it is important to observe these values during experiment on nickel toxicity to obtain more valid results.

Table 2 : Mortality rate of fish during acute nickel exposure for 96 hours. Fish age (at the start of the trial) Nickel exposure concentra- tions (mg L -1 ) Fish mortality (%)
a ǂ:The means with different letters (a & b) in a single row are statistically different at p < 0.05.